Combination Therapy of Immunotoxin and Checkpoint Inhibitor

ABSTRACT

Regional, tumor-targeted, cytotoxic therapy, such as D2C7-immunotoxin (D2C7-IT), not only specifically target and destroy tumor cells, but in the process initiate immune events that promote an in situ vaccine effect. The antitumor effects are amplified by immune checkpoint blockade which engenders a long-term systemic immune response that effectively eliminates all tumor cells.

This invention was made with government support under CA197264 andCA-154291 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

TECHNICAL FIELD OF THE INVENTION

This invention is related to the area of immunotherapy. In particular,it relates to combination regimens for treating tumors and kits andmedicaments for accomplishing them.

BACKGROUND OF THE INVENTION

Glioblastoma is the most dismal malignant brain tumor among all primarybrain and central nervous system tumors. The median survival time forglioblastoma patients with the current standard treatment or even newlydeveloped agents is less than 15 months. Thus, there is an urgent needto develop advanced and efficient therapeutic approaches to improve thepoor survival outlook of glioblastoma patients as well as other tumorsexpressing EGFR receptors.

SUMMARY OF THE INVENTION

According to one aspect of the invention a method is provided fortreating a tumor in a patient. An immunotoxin and an immune checkpointinhibitor are administered to the patient. The immunotoxin comprises asingle chain variable region antibody fused to a PE38 truncatedPseudomonas exotoxin. The single chain variable region antibody hasCDR1, CDR2, and CDR3 regions as shown in SEQ ID NO: 6-11.

According to another aspect of the invention a kit is provided fortreating a tumor. The kit comprises an immunotoxin and an immunecheckpoint inhibitor. The immunotoxin comprises a single chain variableregion antibody fused to a PE38 truncated Pseudomonas exotoxin, whereinthe single chain variable region antibody has CDR1, CDR2, and CDR3regions as shown in SEQ ID NO: 6-11;

These and other embodiments which will be apparent to those of skill inthe art upon reading the specification provide the art with treatmentmethods, regimens, kits, and agents for treating glioblastomas and othertumors expressing epidermal growth factor (EGF) receptors i.e., EGFR andits mutants, e.g., EGFR variant III.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Possible mechanism for combination of regional cytotoxic therapyand immune checkpoint blockade. Cytotoxic therapy may engender targeteddestruction of primary tumors and an ‘in situ vaccine’ effect.Simultaneous blockade of immune checkpoint receptors PD1, PD-L1, TIM-3,LAG-3, and/or CSF-1R may augment the vaccine-mediated immunity. APCs:Antigen Presenting Cells; PD1: Programmed Death molecule 1; PD-L1:Programmed Death-Ligand 1; TIM-3: T cell Immunoglobulin and Mucindomain-3; LAG-3: Lymphocyte Activation Gene-3; CSF-1R: ColonyStimulating Factor 1 Receptor; NK/NKT: natural killer/natural killer Tcells.

FIGS. 2A-2D Flow cytometry analyses of the CT-2A-mD2C7 cell line. (FIG.2A) mouse H-2Kb (MHC class I) expression, (FIG. 2B) mouse H-2Db (MHCclass I) expression, (FIG. 2C) mouse PD-L1 expression, and (FIG. 2D)D2C7-IT target antigen mEGFRvIII expression on CT-2A-mD2C7 cells.

FIG. 3 In vitro cytotoxicity of D2C7-IT on CT-2A-mD2C7 cells. WST1 assaywas utilized to determine the in vitro cytotoxicity of D2C7-IT onCT-2A-mD2C7 cells. The IC₅₀ of D2C7-IT on CT-2A-mD2C7 cells was 0.47ng/ml (lower line) compared to that of the negative control immunotoxin,P588-IT (upper line, IC₅₀>1000 ng/ml).

FIG. 4. Survival of C57BL/6 mice injected intracranially with CT2A-mD2C7cells.

FIGS. 5A-5B. Phenotypic profile of the immune cells populating theCT2A-mD2C7 brain tumor microenvironment. FIG. 5A control brain and FIG.5B CT2A-mD2C7 tumors. (F480lo+F480int+F480hi)=macrophages

FIG. 6. Experimental outline for D2C7-IT, BLZ945 combination therapy.

FIG. 7. Anti-tumor efficacy of D2C7-IT and BLZ945 combination therapyagainst the intracranial CT2A-mD2C7 glioma model.

FIGS. 8A-8B. In vivo efficacy of D2C7-IT+ (αCTLA-4 or αPD-1) mAbcombination therapy in subcutaneous CT2A-mD2C7 glioma-bearing C57BL/6immunocompetent mice. FIG. 8A shows all the treatment groups followed upto Day 35. FIG. 8B shows treatment groups G4-6 followed up to Day 62after initial tumor inoculation. Note that 4 out of 10 and 5 out of 10mice were cured only in treatment groups G5 and G6, respectively.

FIGS. 9A-9B. Tumor rechallenging studies for the cured mice in thecombinational treatment groups (G5 and G6). FIG. 9A. Cured mice werefirst rechallenged with 10⁶ CT2A parental cells on the left flank on Day72. FIG. 9B shows subsequent challenge with 3×10⁵ CT2A-mD2C7 cells inthe brain on Day 126 after the initial CT2A-mD2C7 cell inoculation onthe right flank. Tumors grew in all naïve mice (G0), whereas no tumorgrew in those cured mice (G5 and G6).

FIGS. 10A-10B. In vivo efficacy of D2C7-IT+ (αCTLA-4 or αPD-1) mAbcombination therapy in bilateral subcutaneous CT2A-mD2C7 glioma-bearingC57BL/6 immunocompetent mice. FIG. 10A shows the tumor growth curve forthe right (treated) tumors, which was similar to the previous one-sidemodel. FIG. 10B shows the tumor growth curve for the left (untreated)tumors, which showed that the combinational therapy on the right sidecould also have an antitumor effect on the distant left tumor.

FIGS. 11A-11B. Left tumor volume among groups on Day 35 and Day 43 afterthe initial tumor inoculation. FIG. 11A. On Day 35, compared totreatment group G1, the left tumor growth was significantly delayed inall the other treatment groups, p<0.05 for treatment groups G2, G3, andG4; p<0.01 for treatment groups G5 and G6. FIG. 11B. On Day 43, the lefttumor growth was significantly delayed by the combinational therapycompared to the D2C7-IT monotherapy on the right tumor, p<0.05.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have developed targeted immunotoxins (IT),D2C7-(scdsFv)-PE38KDEL (D2C7-IT), by fusing the single chain variablefragment (scFv) from the D2C7 monoclonal antibody (mAb) with thePseudomonas exotoxin A (PE), optionally fused to KDEL peptide. D2C7-ITreacts with both the wild-type epidermal growth factor receptor (EGFRwt)and the EGFR variant III (EGFRvIII), two proteins that are overexpressedin glioblastoma. The robust antitumor efficacy of D2C7-IT is mediatedthrough PE in orthotopic glioma xenograft models in immunocompromisedmice. In addition to direct tumor cell killing, the immunotoxinmonotherapy induces a secondary antitumor immune response through theengagement of T cells. When the immunotoxin is administered in acombination regimen with an immune checkpoint inhibitor, improved andsynergistic results are observed.

Other moieties which can be attached to the antibodies include thosewhich provide additional beneficial properties. For example, a KDEL(lys-asp-glu-leu) tetra-peptide can be added at the carboxy-terminus ofthe protein to provide retention in the endoplasmic reticulum. Variantssuch as DKEL, RDEL, and KNEL which function similarly can also be used.

Tumors which can be treated are any that react with the D2C7 antibody.These include but are not limited to those in which at least oneEGFRvIII allele is present. These may be found in breast, head and neck,brain, glioblastoma multiforme, astrocytoma, lung, or other tumors. Itmay be desirable to determine the presence of such an allele prior totherapy. This can be done using an oligonucleotide-based technique, suchas PCR, or using an immunological technique, such asimmunohistochemistry. It may be desirable to determine the amount,fraction, ratio, or percentage of cells in the tumor which express EGFRand/or EGFRvIII. The more cells which express EGFR on their surfaces,the more beneficial such antibody therapy is likely to be. Even tumorsthat express little to no EGFRvIII may be treated due to the ability ofthe antibody to bind to wild-type EGFR. Optionally, tumors may be testedprior to treatment for reactivity with D2C7 antibody. The immunotoxinitself could be used as an immunohistochemistry agent, before treatment,during treatment, or after treatment. A secondary reagent could be usedwith the immunotoxin for detection. It could, for example, recognize thePseudomonas component of the immunotoxin.

Immunotoxins can be administered by any technique known in the art.Compartmental delivery may be desirable to avoid cytotoxicity for normaltissues that express EGFR. Suitable compartmental delivery methodsinclude, but are not limited to delivery to the brain, delivery to asurgically created tumor resection cavity, delivery to a natural tumorcyst, and delivery to tumor parenchyma.

Tumors which can be treated by the method of the present invention areany which express epidermal growth factor receptor (EGFR), whether wildtype, EGFRvIII, or other variants. Preferably the tumor expresses thereceptor in amounts far exceeding expression by normal tissues. Themechanism of high level expression may be by genetic amplification,other alterations, whether genetic or epigenetic or post translationalmodifications. Exemplary tumors which can be treated include withoutlimitation: malignant gliomas, breast cancer, head and neck squamouscell carcinoma, lung cancer.

Blockade of T cell immune checkpoint receptors, can be performed againstany such targets, including but not limited to PD-1, PD-L1, TIM-3,LAG-3, CTLA-4, and CSF-1R and combinations of such checkpointinhibitors. The immune checkpoint receptors may be on tumor cells orimmune cells such as T cells, monocytes, microglia, and macrophages,without limitation. The agents which assert immune checkpoint blockademay be small chemical entities or polymers, antibodies, antibodyfragments, single chain antibodies or other antibody constructs,including but not limited to bispecific antibodies and diabodies.

Immune checkpoint inhibitors which may be used according to theinvention include any that disrupt the inhibitory interaction ofcytotoxic T cells and tumor cells. These include but are not limited toanti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA4 antibody, anti-LAG-3antibody, anti-TIM-3 antibody. The inhibitor need not be an antibody,but can be a small molecule or other polymer. If the inhibitor is anantibody it can be a polyclonal, monoclonal, fragment, single chain, orother antibody variant construct. Inhibitors may target any immunecheckpoint known in the art, including but not limited to, CTLA-4, PDL1,PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, CSF-1R, VISTA,KIR, 2B4, CD160, CGEN-15049, CHK 1, CHK2, IDO, A2aR, and the B-7 familyof ligands. Combinations of inhibitors for a single target immunecheckpoint or different inhibitors for different immune checkpoints maybe used.

Examples of inhibitors of CSF-1R which may be used in the combinationtherapy with the immunotoxin include, without limitation, the followingagents which are in clinical development: PLX3397, PLX486, RG7155,AMG820, ARRY-382, FPA008, IMC-CS4, JNJ-40346527, and MCS110.

The immune checkpoint inhibitor may be administered at the same time,before, or after the immunotoxin. Typically the two agents will beadministered within 30, 28, 21, 14, 7, 4, 2, or 1 day(s) of each other.The agents may be given repeatedly, either serially or in a cycle offirst and second agents. It may be advantageous but not necessary forthe vaccine to be administered prior to the checkpoint inhibitor. Butthe reverse order may also be used. Priming of a cytotoxic T lymphocyteresponse by the immunotoxin may take from about 5 to about 14 days.Administration of the checkpoint inhibitor may beneficially be commencedduring or after the priming period.

Immune checkpoint inhibitors may be administered by any appropriatemeans known in the art for the particular inhibitor. These includeintravenous, oral, intraperitoneal, sublingual, intrathecal,intracavitary, intramuscularly, and subcutaneously.

Treatment regimens may include, in addition to delivery of theimmunotoxin and immune checkpoint inhibitor(s), surgical removal of thetumor, surgical reduction of the tumor, chemotherapy, biologicaltherapy, radiotherapy. These modalities are standard of care in manydisease states, and the patient need not be denied the standard of care.The immunotoxin and immune checkpoint inhibitor(s) may be administeredbefore, during, or after the standard of care. The immunotoxin andimmune checkpoint inhibitor(s) may be administered after failure of thestandard of care.

Kits may comprise, in a single divided or undivided container, theimmunotoxin or its components or its encoding DNA and the immunecheckpoint inhibitor or combination of immune checkpoint inhibitors.Storage stability may vary between the two agents so separate vesselsmay be used. Optionally one or both agents may be lyophilized or frozen.

Immunotoxins can directly kill cancer cells that express high levels ofthe targeted tumor antigen. Immunotoxin monotherapy can efficiently anddirectly destroy tumor cells expressing targeted epitopes, such asEGFRwt and/or its truncated variant, EGFRvIII, in malignant brain tumorxenograft models in immunocompromised mice. Immunotoxin therapy caninduce a secondary anti-tumor immune response in a mouse model, which isdifferent from the direct killing mechanism and needs the cooperation ofthe immune system. Since malignant brain tumors are always aheterogeneous mass, it is possible that some tumor cells can escape fromthe direct targeted attack of the immunotoxin therapy due to the lack ofepitopes. For this reason, the secondary anti-tumor immune responsestimulated by the immunotoxin may play an important role in eliminatingthose tumor cells not directly targeted.

Recently, several studies successfully demonstrated that tumorregression and significantly improved survival were achieved in murineglioma models by suppressing co-inhibitory molecules, such as CTLA4,CSF-1R, IDO, and PD1. Based on the promising preclinical data, severalclinical trials have started to investigate the utilization of immunecheckpoint inhibitors to treat malignant brain tumors, either asmonotherapy or combinatorial therapy with other anti-tumor agents.

However, malignant gliomas, including glioblastomas, have relatively lowmutation rates, which may generate fewer and subtle tumor antigens,leading to relatively poor basal immunogenicity compared to other tumortypes that respond well to immunotherapies, for example, melanoma andNSCLC. Therefore, a combination of targeted cytotoxic immunotherapy andimmune checkpoint inhibitors may provide a synergistic anti-tumoreffect.

The ideal combinatorial therapy may have a lower dose of targetedcytotoxic immunotherapy to limit its side effects, and achieve long-termanti-tumor immunity. Immunotoxin therapy can efficiently and directlykill cancer cells that express high levels of the targeted antigenthrough its unique cytotoxic mechanism. Cancer cells destroyed bylocalized immunotoxin therapy release tumor antigens and/or otherneoantigens. These antigens can then be presented by the APCs to host Tcells in the local draining lymph nodes, which activate CTLs to migrateand eliminate the remaining or recurrent tumor cells expressing specifictumor antigens at the tumor site. Throughout this process, variousco-inhibitory checkpoint pathways between T cells and APCs and/orbetween T cells and tumor cells can trigger different mechanisms tode-activate T cells, and to adjust the continuation and intensity of theanti-tumor immunity. Immune checkpoint inhibitors, such as anti-CTLA4and anti-PD1 mAbs, can block these immunosuppressive pathways andtherefore augment tumor cell death caused by lymphocytes activated bythe targeted immunotoxin therapy.

We established a subcutaneous mouse CT2A-mD2C7 glioma model in C57BL/6immunocompetent mice with six groups, in which the mice were treated bythe control immunotoxin P588-IT or D2C7-IT, combined with αCTLA4 or αPD1inhibitors after the tumor grew to a certain size. In this in vivosubcutaneous CT2A-mD2C7 glioma model, four doses of the low-dose D2C7-ITbut not αCTLA4 or αPD1 monotherapy, and D2C7-IT+αCTLA-4 or αPD-1combinatorial therapy generated a significant delay in tumor growthcompared to the control immunotoxin P588-IT treatment groups (FIGS. 8Aand 8B). Importantly, complete cures were only observed inD2C7-IT+αCTLA4 (n=4/10) and D2C7-IT+αPD-1 (n=5/10) combinatorial therapygroups (FIGS. 8A and 8B), although D2C7-IT monotherapy could alsosignificantly delay the tumor growth. These results demonstrated thatlow doses of cytotoxic immunotoxin therapy can significantly delay thetumor growth but fail to cure the tumor-bearing mice. Combined withimmune checkpoint inhibitors, cytotoxic immunotoxin therapy can increasethe initial cure rate of low-dose immunotoxin therapy from zero to over40%. Furthermore, all cured mice rejected the mD2C7-negative tumorsprimary subcutaneous rechallenge, whereas tumors grew in untreated naïvemice, suggesting that the combinatorial treatment provided long lastinganti-tumor immunity that even extended to mD2C7-negative parental cells,as well (FIG. 9A). All nine cured mice then rejected the CT2A-mD2C7secondary intracranial rechallenge in the brain, whereas tumors grew inuntreated naïve mice, indicating that the combinatorial treatment alsoprovided long lasting anti-tumor immunity that extended to the remoteimmune-privileged CNS as well (FIG. 9B), in which the central memory Tcells (T_(CM)) might play an important role.

Subsequently, we established a bilateral subcutaneous mouse glioma modelto investigate whether a localized high-dose immunotoxin treatment canprovide a systemic anti-tumor effect on the tumors in the distal region,and whether the combination of immune checkpoint inhibitors can enhancethis systemic anti-tumor immunity induced by the localized immunotoxintherapy. The D2C7-IT monotherapy, D2C7-IT+αCTLA4, and D2C7-IT+αPD1combinatorial therapy led to significant growth delay of the righttumors (P<0.01), and cured 4/10, 6/10, and 5/10 right tumors,respectively (FIG. 10A). Interestingly, in the groups where the righttumors were treated by D2C7-IT or αCTLA-4 or αPD-1 monotherapy orD2C7-IT+(αCTLA-4 or αPD-1) combinatorial therapy, the left untreatedtumors also grew much slower compared to the control group (FIGS. 10Band 11A), which indicates that a high dose of localized D2C7-ITmonotherapy can achieve a similar anti-tumor immunity on the leftuntreated tumors compared to the systemic immune checkpoint inhibitormonotherapy. Furthermore, the combination therapy in the right tumorsled to the most significantly delayed growth of the left untreatedtumors in the mice (FIGS. 10B and 11B), which suggests that immunecheckpoint inhibitors can enhance the anti-tumor immunity induced by thelocalized immunotoxin therapy to restrict the tumor growth in the remotearea.

We have demonstrated that the intratumoral delivery of D2C7-IT inducessecondary anti-tumor immunity, which destroys not only mD2C7-expressingtumor cells, but also tumor cells not expressing mD2C7 at the systemiclevel. A combination of D2C7 immunotoxin with immune checkpointinhibitors can enhance this immunotoxin-induced anti-tumor immunity toachieve a synergistic long-term anti-tumor effect.

The above disclosure generally describes the present invention. Allreferences disclosed herein are expressly incorporated by reference. Amore complete understanding can be obtained by reference to thefollowing specific examples which are provided herein for purposes ofillustration only, and are not intended to limit the scope of theinvention.

Example 1

We established a mouse glioma line, CT-2A-mD2C7, overexpressing theD2C7-IT antigen mouse EGFRvIII (mEGFRvIII). The reactivity andtherapeutic efficacy of D2C7-IT against CT-2A-mD2C7 cells was determinedby flow cytometry and in vitro cytotoxicity assays (WST1), respectively.CT-2A-mD2C7 cells were further analyzed for MHC class I and PD-L1expression by flow cytometry. In vivo efficacy of D2C7-IT or αCTLA-4 orαPD-1 monotherapy or D2C7-IT+αCTLA-4 or D2C7-IT+αPD-1 combinationtherapy was evaluated in subcutaneous CT-2A-mD2C7 glioma-bearing C57BL/6immunocompetent mice.

WST-1 is a reagent for measuring cell proliferation. It is used for thenonradioactive, spectrophotometric quantification of cell proliferationand viability in cell populations. The assay is based on the cleavage ofthe tetrazolium salt WST-1 to formazan by cellular mitochondrialdehydrogenases. Expansion in the number of viable cells results in anincrease in the activity of the mitochondrial dehydrogenases, which inturn leads to increase in the amount of formazan dye formed. Theformazan dye produced by viable cells can be quantified by measuring theabsorbance at λ=440 nm.

Example 2

Flow cytometry analysis confirmed the specific binding ability of D2C7monoclonal antibody to the CT-2A-mD2C7 cells (FIG. 2D). Flow cytometryalso demonstrated a high expression of both MHC class I molecules (FIGS.2A and 2B) and PD-L1 (FIG. 2C) on tumor cell surface. D2C7-IT was highlycytotoxic (IC50=0.47 ng/mL) against CT-2A-mD2C7 cells in in vitro WST1cytotoxicity assay (FIG. 3).

Example 3

Construction, expression, and purification of D2C7-(scdsFv)-PE38KDELimmunotoxin. The carboxyl terminus of the D2C7 V_(H) domain wasconnected to the amino terminus of the V_(L) domain by a 15-amino-acidpeptide (Gly₄Ser)₃ linker. In order to obtain a stable IT, it isessential to ensure that during renaturation V_(H) is positioned nearV_(L). This was achieved by mutating a single key residue in each chainto cysteine, for the stabilizing disulfide bond to form. On the basis ofpredictions using molecular modeling and empirical data with otherdsFv-recombinant ITs, we chose one amino acid in each chain to mutate tocysteine. These are residues 44 in the framework region 2 (FR2) of V_(H)and 100 in the FR4 of V_(L) (according to the Kabat numbering). Thus, weprepared an Fv that contains both a peptide linker and a disulfide bondgenerated by cysteine residues that replace Ser44 of V_(H) and Gly100 ofV_(L). The D2C7 (scdsFv) PCR fragment was then fused to DNA for domainsII and III of Pseudomonas exotoxin A. The version of Pseudomonasexotoxin A used here, PE38KDEL, has a modified C terminus whichincreases its intracellular retention, in turn enhancing itscytotoxicity. The D2C7-(scdsFv)-PE38KDEL was expressed in E. coli underthe control of T7 promoter and harvested as inclusion bodies.

Example 4

Targeting tumor and tumor-associated macrophages for glioblastomatherapy: We evaluated the ability of D2C7-IT and BLZ945 combinationtreatment to function synergistically and produce an effective antitumorresponse in immunocompetent glioblastoma mouse models.

Intracranial growth curve for CT2A-mD2C7 in C57BL/6 mice: To determinethe time course of CT2A-mD2C7 intracranial tumor growth, 3×10⁵ cells/3μl were implanted into 9 female C57BL/6 mice, and a survival curve wasplotted (FIG. 4). The CT2A-mD2C7 survival curve demonstrated that 100%death occurred at day 25 post-tumor implantation. Based on our previousstudies, post-tumor implantation day 8 was chosen as the optimal day toinitiate D2C7-IT infusion.

Phenotypic profile of the immune cells populating the CT2A-mD2C7 braintumor microenvironment: To characterize the immune cell phenotype ofintracranial CT2A-mD2C7 tumors, C57BL/6 immunocompetent mice wereimplanted with 3×10⁵ tumor cells. The mice were followed to assess tumordevelopment and were euthanized when they became moribund. Uponeuthanization, the brains were harvested and the tumors were analyzedfor infiltrating immune cells by flow cytometry. Cells isolated fromnaïve C57BL/6 mice were used as the control. The primary cell types inthe normal brain were microglia (80%), macrophages(F480lo+F480int+F480hi=11%), and T cells (5%) (FIG. 5A). However, therewas a significant change in the percentage of microglia (8%),macrophages (F480lo+F480int+F480hi=63%), and T cells (19%) intumor-bearing mice (FIG. 5B).

Anti-tumor efficacy of D2C7-IT and BLZ945 combination therapy againstthe intracranial CT2A-mD2C7 glioma model: The experimental outline forD2C7-IT+BLZ945 combination therapy against the CT2A-mD2C7 cell line isshown in FIG. 6. The in vivo efficacy of D2C7-IT or BLZ945 monotherapyor D2C7-IT+BLZ945 combination therapy was evaluated in intracranialCT2A-mD2C7 glioma-bearing C57BL/6 immunocompetent mice (FIG. 7). D2C7-IT(0.018 μg total dose) was infused by an osmotic pump viaconvection-enhanced delivery (CED) for 72 hours from Day 8 to Day 11post-tumor inoculation. BLZ945 (200 mg/kg) was delivered once dailythrough oral gavage on day 7 and on days 12-25. The survival curves formice treated with the vehicle control and with BLZ945 monotherapy lookedsimilar (Median Survival=22-24 days, FIG. 7). At a total dose of 0.018μg, D2C7-IT monotherapy extended the median survival to 42 days (FIG.7). The median survival for the D2C7-IT+BLZ945 combination therapy groupwas 53 days (FIG. 7). Significantly, complete cures were observed onlyin the combination therapy group (2/6 mice). The preliminary datasuggests that glioblastoma patients will benefit from D2C7-IT and BLZ945combination therapy.

Example 5 In Vivo Efficacy of D2C7-IT+(Anti-CTLA4 or Anti-PD1)Inhibitors Combinatorial Therapy in a Subcutaneous (SC) CT2A-mD2C7Glioma Model.

In previous pilot studies, we observed that the subcutaneousrechallenged mouse glioma allografts were rejected in thoseimmunocompetent mice bearing SC mouse glioma allografts cured by theintratumoral (i.t.) immunotoxin therapy, suggesting that there can be amemory anti-tumor immune response following the SC immunotoxin therapy.This phenomenon was also reported in the SC melanoma mouse model treatedby an immunotoxin targeting IL-13, in which CTLs played a major role inmediating this immunotoxin-induced anti-tumor response, althoughmelanoma is a dramatically different type of tumor compared to malignantgliomas in the CNS. Therefore, it is necessary to establish appropriatemouse glioma models to investigate the secondary immune response,induced by immunotoxins, against glioblastomas, and to determine how toenhance this response by the combinatorial therapy of immune checkpointinhibitors, such as anti-CTLA4 or anti-PD1 antibodies (αCTLA4 or αPD1),in order to achieve a long-lasting remission.

We established a subcutaneous mouse CT2A-mD2C7 glioma model in C57BL/6immunocompetent mice with six groups, in which the mice were treated bythe control immunotoxin P588-IT or D2C7-IT, combined with αCTLA4 or αPD1inhibitors. In this in vivo subcutaneous CT2A-mD2C7 glioma model, fourdoses (every 3 days) of the D2C7-IT (low dose, 1.5 μg per mouse perdose, i.t.) but not αCTLA4 (100 μg per mouse per dose, intraperitoneal[i.p]) or αPD1 (250 μg per mouse per dose, i.p.) monotherapy andD2C7-IT+αCTLA-4 or αPD-1 combinatorial therapy (immune checkpointinhibitors administered on the next day after IT therapy) generated asignificant delay in tumor growth compared to the control immunotoxinP588-IT treatment groups (P<0.01, FIG. 8A). Importantly, complete cureswere only observed in D2C7-IT+αCTLA4 (n=4/10) and D2C7-IT+αPD-1 (n=5/10)combinatorial therapy groups (FIGS. 8A and 8B), although D2C7-ITmonotherapy could also significantly delay the tumor growth.

Example 6

Tumor Rechallenging Studies on the Cured Mice from the D2C7-IT andImmune Checkpoint Inhibitors Combinatorial Treatment Groups

To determine whether those cured mice from D2C7-IT and immune checkpointinhibitors combinatorial treatment groups can recall a protectiveanti-tumor memory immune response, on Day 72 after the initial tumorchallenge, all nine cured mice were then first subcutaneouslyrechallenged (1° SCR) with a dose of 10⁶ CT2A parental cells in the leftflank. All these mice rejected the mD2C7-negative tumors, whereas tumorsgrew in all untreated naïve mice, suggesting that the combinatorialtreatment provided long lasting anti-tumor immunity that extended tomD2C7-negative parental cells as well (FIG. 9A).

To determine whether this protective anti-tumor immunity can protect themice from the tumor rechallenging in a remote immune-privileged region,for example, the brain, all nine cured mice were then intracranially(IC) rechallenged (2° ICR) for a second time on Day 126 (after theinitial subcutaneous tumor challenge) with a dose of 3×10⁵ CT2A-mD2C7cells in the brain. At the end of this study, all surviving mice wereeuthanized for brain histopathologic examination, which did not showtumors in the brains (data not shown). All these mice rejected theCT2A-mD2C7 intracranial (IC) rechallenge (2° ICR), whereas tumors grewin all untreated naïve mice, suggesting that the combinatorial treatmentalso provided long lasting anti-tumor immunity that extended to theremote immune-privileged CNS as well (FIG. 9B).

Example 7 In Vivo Efficacy of D2C7-IT+(Anti-CTLA4 or Anti-PD1)Inhibitors Combinatorial Therapy in a Bilateral Subcutaneous CT2A-mD2C7Glioma Model.

In an in vivo bilateral subcutaneous CT2A-mD2C7 glioma model, tumorcells were inoculated in both sides of the flank simultaneously inC57BL/6 mice, with a high density (3×10⁶ cells) on the right side and alow density (10⁶ cells) on the left side. The larger tumors (on theright) were treated with four doses (every two days) of D2C7-IT orαCTLA4 or αPD1 monotherapy or D2C7-IT+αCTLA4 or D2C7-IT+αPD1 combinationtherapy (immune checkpoint inhibitors administered on the same day ofimmunotoxin therapy), while the left tumors were untreated. The D2C7-ITmonotherapy (high dose, 4.5 μg per mouse per dose, intratumoral),D2C7-IT+αCTLA4 (100 μg per mouse per dose, intraperitoneal), andD2C7-IT+αPD1 (250 μg per mouse per dose, intraperitoneal.) combinatorialtherapies led to significant growth delays of the right tumor (P<0.01),which cured 4/10, 6/10, and 5/10 right tumors, respectively (FIG. 10A).

Interestingly, in the groups where the right tumors were treated withD2C7-IT or αCTLA-4 or αPD-1 monotherapy or D2C7-IT+(αCTLA-4 or αPD-1)combinatorial therapy, the left untreated tumors also grew much slowercompared to the control group (FIGS. 10B and 11A), which indicates thata high dose of localized D2C7-IT monotherapy can achieve a similaranti-tumor immunity in the left untreated tumors compared to thesystemic immune checkpoint inhibitor monotherapy. Furthermore, theD2C7-IT+(αCTLA-4 or αPD-1) combinatorial therapy in the right tumors ledto the most significantly delayed growth of the left untreated tumors inthe mice (FIGS. 10B and 11B), which suggests that immune checkpointinhibitors can enhance the anti-tumor immunity induced by theimmunotoxin to restrict the tumor growth in the remote area.

1. A method of treating a tumor in a patient, comprising: administeringto the patient an immunotoxin comprising a single chain variable regionantibody fused to a PE38 truncated Pseudomonas exotoxin, wherein thesingle chain variable region antibody has CDR1, CDR2, and CDR3 regionsas shown in SEQ ID NO: 6-11; and administering an immune checkpointinhibitor to the patient.
 2. The method of claim 1 wherein the tumor isa malignant glioma.
 3. The method of claim 1 wherein the tumor is breastcancer.
 4. The method of claim 1 wherein the tumor is head and necksquamous cell carcinoma.
 5. The method of claim 1 wherein the tumor islung cancer.
 6. The method of claim 1 wherein the immunotoxin isadministered directly to the tumor.
 7. The method of claim 1 wherein theimmune checkpoint is selected from the group consisting of PD-1, PD-L1,CTLA-4, LAG-3, TIM-3, and CSF-1R.
 8. The method of claim 1 wherein thecheckpoint inhibitor is an anti-PD-1 antibody.
 9. The method of claim 1wherein the checkpoint inhibitor is an anti-PD-L1 antibody.
 10. Themethod of claim 1 wherein the checkpoint inhibitor is an anti-CTLA4antibody.
 11. The method of claim 1 wherein the checkpoint inhibitor isan anti-LAG-3 antibody.
 12. The method of claim 1 wherein the checkpointinhibitor is an anti-TIM-3 antibody.
 13. The method of claim 1 whereinthe checkpoint inhibitor is an anti-CSF-1R antibody.
 14. The method ofclaim 1 wherein the checkpoint inhibitor is a small molecule inhibitorof IDO.
 15. The method of claim 1 wherein the checkpoint inhibitor is asmall molecule inhibitor of CSF-1R.
 16. The method of claim 1 whereinthe immune checkpoint inhibitor is administered within 30 days ofadministering the immunotoxin.
 17. The method of claim 1 wherein theimmune checkpoint inhibitor is administered within 7 days ofadministering the immunotoxin.
 18. The method of claim 1 wherein thePE38 truncated Pseudomonas exotoxin is fused to a KDEL peptide.
 19. Akit for treating a tumor, comprising: an immunotoxin comprising a singlechain variable region antibody fused to a PE38 truncated Pseudomonasexotoxin, wherein the single chain variable region antibody has CDR1,CDR2, and CDR3 regions as shown in SEQ ID NO: 6-11; and an immunecheckpoint inhibitor.
 20. The kit of claim 19 wherein the checkpointinhibitor is an anti-PD-1 antibody.
 21. The kit of claim 19 wherein thecheckpoint inhibitor is an anti-PDL-1 antibody.
 22. The kit of claim 19wherein the checkpoint inhibitor is an anti-CTLA4 antibody.
 23. The kitof claim 19 wherein the checkpoint inhibitor is an anti-LAG-3 antibody.24. The kit of claim 19 wherein the checkpoint inhibitor is ananti-TIM-3 antibody.
 25. The kit of claim 19 wherein the checkpointinhibitor is an anti-CSF-R1 antibody.
 26. The kit of claim 19 whereinthe checkpoint inhibitor is a small molecule inhibitor of IDO.
 27. Thekit of claim 19 wherein the checkpoint inhibitor is a small moleculeinhibitor of CSF-R1.
 28. The kit of claim 19 wherein the PE38 truncatedPseudomonas exotoxin is fused to a KDEL peptide.